: This proposal is designed to identify physiological mechanisms that regulate stem cell maintenance. The investigator will focus on the manipulation of the stroma compartment as a means to understand stem cells, working on the hypothesis that stromal cell lines in vitro mirror in vivo microenvironmental niches that control stem cells. Support for this premise is provided by the investigator's data demonstrating that certain rare stromal cell lines in vitro maintain high levels of stem cells capable of multilineage repopulation of recipient mice. These lines also support human long term culture-initiating cells. Thus, stromal cell lines are powerful tools for the analysis of the mechanisms and molecules that regulate stem cells. Maintenance of stem cells in these cultures is dependent on direct contact to stromal cells and both adhesion molecules and cytokines are important. In particular, the CD44 molecule plays a pivotal role, because different pseudo-ligands, in the form of mAbs, can either enhance or suppress stem cell maintenance. What is missing at our current stage of knowledge is an understanding of how supportive vs. non-supportive stromal cells interact with stem cells, and insights into the regulation of the stromal cell compartment with respect to its ability to support stem cells. The investigator proposes to test the following hypotheses in this application: 1) Stromal cells are intrinsically different in their ability to support stem cells; and 2) this support ability can be modulated extrinsically. These hypotheses will be tested through the identification of unique cell surface markers on stromal cell lines that differ in stem cell support, using a panel of mAb previously generated by the applicant. The contribution of positive and negative regulatory stimuli will be identified in mixing experiments with functionally different stromal cell lines. To characterize extrinsic regulation of stromal cell function, focus will be place on CD44 mediated regulation. The underlying hypothesis to be tested is that regulation of stem cell levels via CD44 targets the stroma compartment in an adhesion-independent event that involves signal transduction. This will be analyzed by: a) determining the target cells for pseudo-ligands and ligands for both human and mouse LTC-IC; b) identifying the physiological ligands; and c) determining the stromal cell programs induced by pseudo ligands and/or ligands. Thus, the proposed studies will define more precisely the mechanisms and the signals important for stem cell maintenance and provide insight into the regulation of the stroma compartment. An understanding of these events will ultimately be beneficial for the treatment of a variety of hematopoietic diseases in man, and will be important for stem cell transplantation and gene therapy.